Abstract
This is the first report of in vitro propagation and alkaloid accumulation in callus cultures of Ceropegia juncea Roxb. a source of “Soma” drug in Ayurvedic medicine. Multiple shoots and callus induction was optimized by studying the influence of auxins [IAA (Indole-3-acetic acid), NAA (2-Naphthalene acetic acid) and 2,4-D (2,4-Dichlorophenoxyacetic acid.)] and cytokinins [BA (6-benzyladenine) and Kin (Kinetin)] alone and in combinations. The best response for multiple shoot induction was obtained in nodal explants on MS medium supplemented with 7.5 μM Kin (8.5 ± 3 shoots per explants). The shoots were rooted on half strength MS (Murashige and Skoog’s) medium fortified with either IAA or NAA (0.5–2.0 μM). The plantlets were transferred directly to the field with 100 % success rate. Supplementation of MS medium with auxins and cytokinins enhanced the growth of callus but inhibited the shoot regeneration in nodal explants. Best callus induction and proliferation observed on MS + 1 μM 2,4-D+5 μM BA. However the maximum cerpegin content (470 μg/g dry weight) was recorded in dried callus derived on MS+10 μM IAA+5 μM BA. Quantitative TLC (Thin layer chromatography) studies of the callus revealed a phytochemical profile similar to that of naturally grown plants. The calli were maintained by subculturing at 4 weeks interval on fresh parent medium over a period of 34 months. The optimized in vitro propagation and callus culture protocol offers the possibilities of using organ/callus culture technique for vegetative propagation and production of cerpegin alkaloid.
Key words: In vitro propagation, Pyridone alkaloid, Cerpegin, Callus, Ceropegia juncea
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References
- Adibatti N.A., Thirugnanasambantham P., Kuilothungan C. A pyridine alkaloid from Ceropegia juncea. Phytochem. 1991;30:2449–2450. doi: 10.1016/0031-9422(91)83683-C. [DOI] [Google Scholar]
- Asolkar L.V., Kakkar K.K., Chakre O.J. IInd supplement to the Glossary of Indian Medicinal plant with Active Principle. Part 1 (A-K) New Delhi: Publication and Information Directorate, CSIR; 1992. p. 193. [Google Scholar]
- Beena M.R., Martin K.P., Kirti P.B. Rapid in vitro propagation of medicinally important Ceropegia candelabrum. Plant Cell, Tiss. Org. Cult. 2003;72:285–289. doi: 10.1023/A:1022395809204. [DOI] [Google Scholar]
- Benjamine B.D., Heble M.R., Chadha M.S. Alkaloid synthesis in tissue culture and regenerated plants of Tylophora indica Merr. (Asclepiadaceae) Z. Pfanzen. Phsiol. 1979;92:72–84. [Google Scholar]
- Duncans D.B. Multiple range and multiple F-test. Biometrics. 1955;11:1–24. doi: 10.2307/3001478. [DOI] [Google Scholar]
- Hariharan M., Sebastian D.P., Benjamin S. Somatic embryogenesis in Leptadenia reticulata Weght and Arn.Pa medicinal plant. Phytomorphol. 2002;52:55–63. [Google Scholar]
- Jagtap A.P., Singh N.P. Asclepiadaceae and Periplocaceae. Fascicles of Flora of India, Pb1. Botanical Survey of India, Kolkata. Fascicle. 1999;24:211–241. [Google Scholar]
- Jain S.K., Defillips R.A. Asclepiadaceae. In: Algonae M. I., editor. Medicinal plants of India. Vol. 1. Michigan, USA: Reference Publications Inc.; 1991. pp. 144–152. [Google Scholar]
- Komalavalli N., Rao M.V. In vitro micropropagation of Gymnema sylvester — A multipurpose medicinal plant. Plant Cell. Tiss. Org. Cult. 2000;61:97–105. doi: 10.1023/A:1006421228598. [DOI] [Google Scholar]
- Manjula S., Job A., Nair G.M. Somatic embryogenesis from leaf derived callus of Tylophora indica (Burm. f.) Merrill. Indian J. Exp. Biol. 2000;38(10):1069–1072. [PubMed] [Google Scholar]
- Martin K.P. Rapid propagation of Holostemon adakodien Schult., a rare medicinal plant, through axillary bud multiplication and indirect organogenesis. Plant Cell Rep. 2002;21:112–117. doi: 10.1007/s00299-002-0483-7. [DOI] [Google Scholar]
- Murashige T., Skoog F.A. Revised medium for rapid growth and bioassays with tobacco tissue cultures. Physiol Plant. 1962;15:473–497. doi: 10.1111/j.1399-3054.1962.tb08052.x. [DOI] [Google Scholar]
- Nikam T.D., Savant R.S. Callus culture and micropropagation of Ceropegia sahyadrica Ans. And Kulk.: An edible starchy tuberous rare asclepiad. Indian J. Plant. Physiol. 2007;12(2):108–114. [Google Scholar]
- Patil V.M. Micropropagation studies in Ceropegia spp. In Vitro Cell Dev. Biol.-Plant. 1998;30(3):240–243. doi: 10.1007/BF02822714. [DOI] [Google Scholar]
- Ramawat K.G., Merillon J.M. Biotechnology — Secondary Metabolites. Delhi. India: Mohan Primlani for Oxford and IBH publishing.; 1999. pp. 193–218. [Google Scholar]
- Roy A.T., Koutoulis A., De D.N. Cell suspension culture and plant regeneration in the latex-producing plant, Calotropis gigantea (Linn.) R. Br. Plant Cell, Tiss Org. Cult. 2000;63:15–22. doi: 10.1023/A:1006497220647. [DOI] [Google Scholar]
- Sarasan V., Soniya E.V., Nair G.M. Regeneration of Indian Sarsaparillsa, Hemidesmus indiculs R.Br., through organogenesis and somatic embryogenesis. Indian J. Exp. Biol. 1994;32:284–287. [Google Scholar]
- Sreekumar S., Seeni S., Pushpangadan P. Micropropagation of Hemidesmus indicus for cultivation and production of 2-hydroxy 4-methoxy benzaldehyde. Plant Cell Tiss. Org. Cult. 2000;62:211–218. doi: 10.1023/A:1006486817203. [DOI] [Google Scholar]
- Sudha C.G., Krishnan P.N., Pushpangadan P. In vitro propagation of Holostemma annulare (Roxb.) K. Schum., a rare medicinal plant. In Vitro Cell. Dev. Biol. Plant. 1998;33:57–63. [Google Scholar]
- Sukumar E., Gopal H., Rao R.B., et al. Pharmacological actions of cerpegin, a novel pyridine alkaloid from Ceropegia juncea. Fitoterapia. 1995;66:403–406. [Google Scholar]
- Thorpe T.A. History of plant tissue culture. Mol. Biotechnol. 2007;37:169–180. doi: 10.1007/s12033-007-0031-3. [DOI] [PubMed] [Google Scholar]
- Wilson K.J., Mahlberg P.G. Investigation of laticifer differentiation in tissue cultures derived from Asclepias syriaca. Ann. Bot. 1977;41:1049–1054. [Google Scholar]